We aim to elucidate the molecular mechanisms that control the assembly of viruses with the ultimate goals of defining targets for antiviral compounds, characterizing viral antigenicity, and establishing precedents for understanding the assembly of macromolecular complexes in general. Our research focuses on the large-scale conformational changes that accompany capsid maturation and on the interaction of viruses with host cells. To this end, we pursue five subprojects. (1) Hepatitis B Virus Capsid Assembly. We have been studying the HBV capsid protein which presents both core antigen (capsids) and e-antigen (unassembled protein) epitopes. After first showing that capsid protein self-assembles from dimers into particles of two different sizes, in 1997 we calculated a cryo-EM density map in which much of the secondary structure was visible, including the 4-helix bundle that forms the dimerization motif. Our subsequent research helped delineate the path of the polypeptide chain. We are now investigating the antigenic diversity of HBV capsids and the basis for e-antigenicity. New Results. We performed cryo-EM and molecular modeling of Fab-labelled capsids to characterize the binding of two more monoclonal antibodies, bringing the total to six. Their epitopes are found in two regions of the capsid surface. The five that cluster around the spike tip are all distinct, albeit overlapping. E-antigen dimers were found to be capable of assembly into capsids albeit less readily than c-antigen dimers. Conclusions. An extensive range of antigens is generated on the capsid spike by a simple motif of two helix-loop-helix elements. The e-antigen dimer is distorted relative to the c-antigen dimer, affecting both its assembly and antigenicity properties. (2) Capsid Assembly and Maturation of Herpesviruses. Herpesviruses constitute an extensive family of large complex DNA viruses. Eight members cause diseases in humans, including skin diseases. In studying herpesvirus capsid assembly, we first defined its molecular anatomy and then characterized the roles of its six major proteins. These roles are distinct from any other known animal virus but resemble those found in DNA bacteriophages. One major finding was our discovery of the HSV procapsid - a short-lived precursor that differs radically from the mature capsid in structure, stability, and composition. Maturation of the procapsid. controlled by the viral protease, is a target for antiviral drugs. We have visualized this dynamic process by time-resolved cryo-EM. New Results. Over the past year, we completed three projects that have been published or submitted for publication: (i) Determination of the complete three-dimensional structure of the virion by cryo-electron tomography: in particular, we quantitated the viral complement of glycoproteins and demonstrated that the tegument is asymmetric. (ii) We determined the 3D structure of the UL6 portal protein which is located at one vertex of the capsid and represents the channel through which DNA enters the capsid. (iii) We demonstrated that the capsid of an oyster virus has typical herpesvirus architecture despite a totally divergent genome sequence. (3) Assembly and Maturation of Bacteriophage Capsids. Our primary interest in phage capsid assembly lies in the monumental conformational changes that accompany their maturation. These changes are irreversible, large in scale, and stringently controlled, and afford unique opportunities for insight into regulatory conformational changes in protein complexes. We study maturation of several phages to exploit expedient aspects of each system. New Results. To relate structural changes with the energetics of HK97 capsid maturation, we used calorimetry in conjunction with cryo-EM to compare each distinct structural state. The main finding, currently being prepared for publication, is that the network of autocatalytic crosslinks that form between neighboring HK97 subunits not only drive maturation but also confer an essential stabilization. The packing of encapsidated T4 DNA was studied by cryo-EM of mutants and of systematically perturbed capsids. These observations support a spool model, as previously observed in T7, but with the orientation of the T4 spool relative to the portal axis differing according to the prolateness of the capsid, probably reflecting a minimized energy penalty from DNA bending. (4) Cell Entry of Poliovirus. Lacking a lipoprotein envelope, the poliovirus capsid must interact directly with the host cell membrane to effect entry and initiate infection. In previous work, we characterized two aspects of this interaction: (i) the binding of the endodomain of the poliovirus receptor to poliovirus; and (ii) the transition from the 160S state to the 135S state that follows receptor interaction and may be simulated by brief heating. New Results. Over the past year, we have brought to a conclusion more detailed investigations of these reactions. Specifically, we have (i) refined our "tectonic" account of rigid-body movements of the capsid proteins in the 160S-135S transition and localized the externalized N-terminus of VP1; and (ii) compared glycosylated and deglycosylated capsid-bound receptors at twice the previous resolution. (5) Electron Microscopic Studies of HIV- and AIDS-related Proteins. We participate in the NIH Targeted Antiviral Program by pursuing projects in which our expertise in EM-based analysis may be applied. The main thrust of our IATAP work has shifted towards applications of electron tomography. Results. To this end, we completed and published a tomographic analysis of herpes simplex virus (see above) and went on to investigate the cell entry process of mutant and wildtype HIV. Infected cultured cells releasing progeny virions were fixed and embedded and thin sections (100 nm) were cut, stained, and analyzed by tomography. In this ongoing investigation, we are attempting to identify the aspect of "budding' that is impaired in a mutant in which the P6 domain, the terminal domain of the Gag polyprotein has been deleted.